Abstract
Purpose :
After stromal injury, the release of pro-inflammatory cytokines and growth factors typically results in activation of quiescent keratocytes to migratory fibroblast and/or fibrotic myofibroblast phenotypes. To our knowledge, there has not been a comprehensive analysis of the changes in gene expression that underlie these distinct wound healing phenotypes. The goal of this study is to identify transcriptional differences and curate characteristic gene expression profiles for cultured corneal keratocytes, fibroblasts, and myofibroblasts.
Methods :
Primary rabbit corneal keratocytes were cultured for 5 days in serum-free media (SF), serum-containing media (S+), or SF with TGF-β1, to produce keratocytes, fibroblasts, and myofibroblasts, respectively. Four samples of total RNA were collected for each treatment group using the BioRad Aurum Total RNA Mini Kit, and two experimental replicates were sent to Novogene for bulk RNA-sequencing. Subsequent bioinformatics analysis included gene expression quantification, differential expression (DE), and functional analysis.
Results :
Overall, there were 7693 significant (Padj<0.05 and log2(FoldChange)>1) DE genes between keratocytes and fibroblasts, 4430 between keratocytes and myofibroblasts, and 5704 between fibroblasts and myofibroblasts. When comparing S+ and TGF-β1 conditions to SF (control), genes characteristic of a quiescent keratocyte phenotype were generally downregulated (e.g. KERA and ALDH1A1). In S+ conditions, initial analysis revealed upregulation of genes associated with DNA replication, metabolism, and ECM-receptor interactions, and downregulation of cell-cell adhesion markers. There was also DE of genes associated with PI3K-Akt and Rap1 signaling. In TGF-β1 conditions, we observed upregulation of genes associated with focal adhesion formation and ECM remodeling. There was also DE of genes associated with PI3K-Akt, JAK-STAT, MAPK, PPAR, Rap1, and TNF signaling pathways. Additionally, for genes related to growth factor activity, there was an upregulation of TGF-β and PDGF family genes and a downregulation of FGF genes.
Conclusions :
These data demonstrate that there are distinct gene expression profiles for cultured keratocytes, fibroblasts, and myofibroblasts. Further analysis of these transcriptomes may lead to identification of new gene markers/targets and discovery of novel signaling pathways associated with these phenotypes.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.